Investigating the Role of Epiregulin During Human Intestinal Development

项目摘要 肠干细胞(ISCs)负责维持和再生肠上皮，以及 驻留在一个称为隐窝的域中，由组成干细胞的物理和生化线索支持 利基市场。尽管ISCs在人类肠道发育、体内平衡和疾病中发挥着至关重要的作用，但 人们对构成人类ISC生态位的信号线索知之甚少。通过分析单细胞RNA 从怀孕后大约7到21周的人十二指肠样本中提取的测序数据，我们 确定了表皮生长因子(EGF)，这是通常用来创造人工体外利基的 对于生长肠道类器官，是在远离干细胞的绒毛分化细胞上表达的 域。我们还鉴定了另一种EGF家族成员EpiRegin(Ereg)，它高度富含在 干细胞结构域和空间位置已确认到地穴。功能实验表明，培养 Ereg或EGF中分离的肠上皮(称为肠样细胞)导致显著不同的细胞 结果和空间组织。根据这些数据，这项提案的首要目标是确定 ERG作为一种新的肠道干细胞生态位信号的功能作用。我们将检验埃雷格的假设， 而不是EGF，是肠道干细胞稳态的关键生理相关生长因子，并在 在人类肠道发育中起着关键作用。我们将通过以下具体目标来检验这一假设：1) 阐明EGF和Ereg作用于人ISCs的机制；2)评估Ereg在 体外和异种移植后人体肠道发育的复杂模型。

PROJECT ABSTRACT Intestinal Stem Cells (ISCs) are responsible for maintenance and regeneration of the intestinal epithelium, and reside in a domain called the crypt, supported by physical and biochemical cues that make up the stem cell niche. Despite the vital role ISCs play in human intestinal development, homeostasis and disease, the signaling cues that make up the human ISC niche are poorly understood. By analyzing single cell RNA sequencing data from human duodenal samples spanning approximately 7-to-21-week post conception, we determined that Epidermal Growth Factor (EGF), which is commonly used to create an artificial in vitro niche for growing intestinal organoids, was expressed on the differentiated cells of the villus, far from the stem cell domain. We also identified another EGF family member, Epiregulin (EREG), which was highly enriched in the stem cell domain and spatial location confirmed to the crypt. Functional experiments suggest that culturing isolated intestinal epithelium (known as enteroids) in EREG or EGF lead to dramatically different cellular outcomes and spatial organization. Based on this data, the overarching goal of this proposal is to determine the functional role of EREG as a novel intestinal stem cell niche cue. We will test the hypothesis that EREG, and not EGF, is a critical physiologically relevant growth factor for intestinal stem cell homeostasis and plays a key role in human intestinal development. We will test this hypothesis through the following specific aims: 1) Elucidate the mechanism by which EGF and EREG act on human ISCs; 2) Assess the role of EREG in a complex model of the developing human intestine in vitro and following xenotransplantation.